We are engaged in experimental studies of the mechanism of protein folding based on characterizing non-covalent intermediates that are observed in fast kinetic studies of the reversible unfolding transitions of small proteins. Our recent work indicates that the kinetic mechanism of refolding of ribonuclease A (RNase A, disulfide bonds intact) can be represented by a 3-species model U1 reversible yields slow U2 reversible yields fast N, where U1 and U2 are two forms of the heat-unfolded protein with very different rates of refolding, and N is the native enzyme. Currently we are working on final proofs of the correctness of this model for RNase A, of testing its generality when applied to other small proteins, and of trying to learn the nature of the U1 reversible yields U2 reaction. We are also trying to characterize a second intermediate that is observed transiently in unfolding and apparently lies between U2 and N on the pathway of folding. We also are starting to relate the kinetic mechanisms of the thermal and of the guanidine-induced unfolding transitions of RNase A. We are using proton NMR to characterize the folding that can occur in U1 before the U1 reversible yields U2 reaction takes place, in conditions where refolding is slow (pH2, 10 degrees). Finally we are characterizing the concentration-dependent, H exchange kinetics of 3H- labelled S-peptide in RNase S as a model system in which to analyze local unfolding. BIBLIOGRAPHIC REFERENCES: Tandem Genetic Duplications: "On the mechanism of tandem genetic duplications in phage lambda". S.W. Emmons, V. MacCosham and R.L. Baldwin. (1975). J. Mol. Biol. 95, 83-89. Mechanism of Protein Folding: "Intermediates in protein folding reactions and the mechanism of protein folding", R.L. Baldwin (1975). Ann. Rev. Biochem. 44, 453-475.